The present invention relates to peptides derived from protein regions or domains referred to herein as heptad repeat regions. In particular, the invention relates to a peptide referred to herein as DP178 or, alternatively, as T20. DP178 corresponds to amino acid residues 638 to 673 of the HIV-1LAI transmembrane protein (TM) gp41 which correspond to a heptad repeat region referred to as HR2. The invention also relates to portions or analogs of DP178 which exhibit anti-membrane fusion capability, antiviral activity (for example the ability to inhibit HIV transmission to uninfected CD4+ cells) and/or an ability to modulate intracellular processes involving coiled-coil peptide structures.
The present invention also releates to a peptide referred to herein as DP107 or, alternatively, as T21. DP107 corresponds to amino acid residues 558 to 595 of the HIV-1LAI transmembrane protein gp41 which correspond to a heptad region referred to as HR1. The invention also relates to portions or analogs of DP107 which exhibit anti-membrane fusion capability, antiviral activity (for example the ability to inhibit HIV transmission to uninfected CD4+ cells) and/or an ability to modulate intracellular process involving coiled-coil peptide structures.
The invention further relates to other DP178-like and DP107-like peptides derived, e.g., from other species of virus, and which correspond to HR1 and HR2 regions analogous to the HR1 and HR2 regions of the HIV-1 transmembrane protein gp41. Such peptides include, e.g., peptides derived from amino acid sequences of HR1 and HR2 regions of the respiratory syncytial virus (RSV) F1 fusion protein (F1-protein) which are described herein.
As described herein, the HR1 and HR2 regions of proteins such as HIV gp41 and the RSV F1-protein interact (non-covalently) with each other and/or with peptides derived therefrom (such as T20 and T21). This interaction is required for normal infectivity of viruses such as RSV and HIV.
The present invention therefore additionally relates to methods for identifying compounds, including small molecule compounds, that disrupt the interaction between DP178 and DP107 and/or between DP107-like and DP178-like peptides. In one embodiment, such methods relate to identification and utilization of modified DP178, DP178-like, DP107 and DP107-like peptides and peptide pairs that interact with each other at a lower affinity than the affinity exhibited by corresponding xe2x80x9cparentxe2x80x9d or xe2x80x9cnativexe2x80x9d peptides. Further, the invention relates to the use of DP178, DP178 portions, DP107, DP107 portions and/or analogs and other modulators, including small molecule modulators, of DP178/DP107, DP178-like/DP107-like or HR1/HR2 interactions as antifusogenic or antiviral compounds or as inhibitors of intracellular events involving coiled-coil peptide structures.
The invention is demonstrated, first, by way of an Example, wherein DP178 and a peptide whose sequence is homologous to DP178 are each shown to be potent, non-cytotoxic inhibitors of HIV-1 transfer to uninfected CD4+ cells. The invention is further demonstrated by Examples wherein peptides having structural and/or amino acid motif similarity to DP107 and DP178 are identified in a variety of viral and nonviral organisms, including RSV, and in Examples wherein a number of such identified peptides derived from several different viral systems are demonstrated to exhibit antiviral activity. The invention is still further demonstrated by way of other Examples wherein other DP178-like and DP107-like peptides are identified that interact with their corresponding HR1 and HR2 domains with a lower affinity than the affinity exhibited by the native DP178 or DP107 peptide from which they are derived. The invention is still further demonstrated by way of an additional Example wherein DP178-like and DP107-like peptides from the RSV F1-protein are identified and structural studies are carried out demonstrating that these peptides associate non-covalently to form the coiled-coil structure typical of an HR1/HR2 interaction.
Membrane fusion is a ubiquitous cell biological process (for a review, see White, J. M., 1992, Science 258:917-924). Fusion events which mediate cellular housekeeping functions, such as endocytosis, constitutive secretion, and recycling of membrane components, occur continuously in all eukaryotic cells.
Additional fusion events occur in specialized cells. Intracellularly, for example, fusion events are involved in such processes as occur in regulated exocytosis of hormones, enzymes and neurotransmitters. Intercellularly, such fusion events feature prominently in, for example, sperm-egg fusion and myoblast fusion.
Fusion events are also associated with disease states. For example, fusion events are involved in the formation of giant cells during inflammatory reactions, the entry of all enveloped viruses into cells, and, in the case of human immunodeficiency virus (HIV), for example, are responsible for the virally induced cell-cell fusion which leads to cell death.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4+ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed of capsid proteins, that contains the viral RNA genome and those enzymes required for early replicative events. Myristylated Gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane enveloped derived from the infected cell membrane. The HIV enveloped surface glycoproteins are synthesized as a single 160 Kd precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD-4+ cells because the CD-4 cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD-4+ receptor molecules (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348) and thus explains HIV""s tropism for CD-4+ cells, while gp41 anchors the enveloped glycoprotein complex in the viral membrane.
HIV infection is pandemic and HIV associated diseases represent a major world health problem. Although considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H. et al., 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD-4+ T-cells by some HIV-1 strains (Smith, D. H. et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD-4 (Daar, E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD-4 clinical trials have produced inconclusive results (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific secondary processing of certain viral proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 enveloped proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. To this end, several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al., WO 92/22,654; Shafferman, A., WO 91/09,872; Formoso, C. et al., WO 90/07,119. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, a truly effective, non-toxic treatment is still needed.
The present invention relates, first, to DP178, a 36-amino acid synthetic peptide, also referred to herein as T20, corresponding to amino acids 638 to 673 of the transmembrane protein (TM) gp41 from the HIV-1 isolate LAI (HIV-1LAI), which exhibits potent anti-HIV-1 activity. The gp41 region from which DP178 is derived is referred to herein as HR2.
The invention further relates to those portions and analogs of DP178 which also show such antiviral activity, and/or show anti-membrane fusion capability, or an ability to modulate intracellular processes involving coiled-coil peptide structures. The term xe2x80x9cDP178 analogxe2x80x9d refers to a peptide which contains an amino acid sequence corresponding to the DP178 peptide sequence present within the gp41 protein of HIV-1LAI, but found in viruses and/or organisms other than HIV-1LAI Such DP178 analog peptides may, therefore, correspond to DP178-like amino acid sequences present in other viruses, such as, for example, enveloped viruses, such as RSV and other retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such analogous DP178 peptides may also correspond to DP178-like amino acid sequences present in nonviral organisms. For ease of discussion, the regions of other proteins (i.e., proteins from viruses and/or organisms other than HIV-1LAI) from which such DP178 analog peptides are derived are referred to herein as xe2x80x9cHR2 regions.xe2x80x9d
The invention further relates to DP107, a peptide, which is also referred to herein as T21, corresponding to amino acids 558-595 of the HIV-1LAI transmembrane protein (TM) gp41. The gp41 region from which DP107 is derived is referred to herein as HR1. The invention also relates to those portions and analogs of DP107 which that also show antiviral activity, and/or show anti-membrane fusion capability, or an ability to modulate intracellular processes involving coiled-coil peptide structures. The term xe2x80x9cDP107 analogxe2x80x9d as used herein refers to a peptide which contains an amino acid sequence corresponding to the DP107 peptide sequence present within the gp41 protein of HIV-1LAI, but found in viruses and organisms other than HIV-1LAI. Such DP107 analog peptides may, therefore, correspond to DP107-like amino acid sequences present in other viruses, such as, for for example, enveloped viruses, such as retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such DP107 analog peptides may also correspond to DP107-like amino acid sequences present in nonviral organisms. For ease of discussion, the regions of other proteins (i.e., proteins from viruses and/or organisms other than HIV-1LAI) from which such DP107 analog peptides are derived are referred to herein as xe2x80x9cHR1 regions.xe2x80x9d
Further, the peptides of the invention include DP107 analog and DP178 analog peptides having amino acid sequences of HR1 and HR2 domains, respectively, from other proteins such as from other viral proteins. Such xe2x80x9cDP107-likexe2x80x9d and xe2x80x9cDP178-likexe2x80x9d are readily recognized or identified by the 107xc3x97178xc3x974, ALLMOTI5 and/or PLZIP search motifs described herein.
The peptides of the invention may, for example, exhibit antifusogenic activity, antiviral activity, and/or may have the ability to modulate intracellular processes which involve coiled-coil peptide structures. With respect to the antiviral activity of the peptides of the invention, such an antiviral activity includes, but is not limited to the inhibition of HIV transmission to uninfected CD-4+ cells. Additionally, the antifusogenic capability, antiviral activity or intracellular modulatory activity of the peptides of the invention merely requires the presence of the peptides of the invention, and, specifically, does not require the stimulation of a host immune response directed against such peptides.
The peptides of the invention may be used, for example, as inhibitors of membrane fusion-asociated events, such as, for example, the inhibition of human and non-human retroviral, especially HIV, transmission to uninfected cells. It is further contemplated that the peptides of the invention may be used as modulators of intracellular events involving coiled-coil peptide structures.
The peptides of the invention may, alternatively, be used to identify compounds, including small molecule compounds, which may themselves exhibit antifusogenic, antiviral, or intracellular modulatory activity. Additional uses include, for example, the use of the peptides of the invention as organism or viral type and/or subtype-specific diagnostic tools.
The terms xe2x80x9cantifusogenicxe2x80x9d and xe2x80x9canti-membrane fusionxe2x80x9d, as used herein, refer to an agent""s ability to inhibit or reduce the level of membrane fusion events between two or more moieties relative to the level of membrane fusion which occurs between said moieties in the absence of the peptide. The moieties may be, for example, cell membranes or viral structures, such as viral envelopes or pili. The term xe2x80x9cantiviralxe2x80x9d, as used herein, refers to the compound""s ability to inhibit viral infection of cells, via, for example, cell-cell fusion or free virus infection. Such infection may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving a viral structure and a cellular structure (e.g., such as the fusion of a viral pilus and bacterial membrane during bacterial conjugation). It is also contemplated that the peptides of the invention may exhibit the ability to modulate intracellular events involving coiled-coil peptide structures. xe2x80x9cModulatexe2x80x9d, as used herein, refers to a stimulatory or inhibitory effect on the intracellular process of interest relative to the level or activity of such a process in the absence of a peptide of the invention.
Embodiments of the invention are demonstrated below wherein an extremely low concentration of DP178 (SEQ ID NO:15), and very low concentrations of a DP178 homolog (SEQ ID NO:1357) are shown to be potent inhibitors of HIV-1 mediated CD-4+ cell-cell fusion (i.e., syncytium formation) and infection of CD-4+ cells by cell-free virus. Further, it is shown that DP178 (SEQ ID NO:15) is not toxic to cells, even at concentrations 3 logs higher than the inhibitory DP-178 (SEQ ID NO:15) concentration.
The present invention is based, in part, on the surprising discovery that the DP107 and DP178 domains (i.e., the HR1 and HR2 domains) of the HIV gp41 protein non-covalently complex with each other, and that their interaction is required for the normal infectivity of the virus. This discovery is described in the Example presented, below, in Section 8. The invention, therefore, further relates to methods for identifying antifusogenic, including antiviral, compounds that disrupt the interaction between DP107 and DP178, and/or between DP107-like and DP178-like peptides.
Additional embodiments of the invention (specifically, the Examples presented in Sections 9 and 10, below) are demonstrated, below, wherein peptides having structural and/or amino acid motif similarity to DP107 and DP178 are identified from a variety of sources, and search motifs for their identification are described. Further, Examples (e.g., in Section 11) are presented wherein a number of the peptides of the invention are demonstrated exhibit substantial antiviral activity or activity predictive of antiviral activity. Further still, an additional example is presented, in Section 14, wherein additional DP107-like and DP178-like peptides are identified that correspond to HR1 and HR2 domains of the respiratory syncytial virus (RSV) F1-protein and are demonstrated to have properties characteristic of the DP107-like and DP178-like peptides of the invention. In particular, the peptides described in Section 14, below, are shown to associate non-covalently in solution to form a coiled-coil structure characteristic of HR1/HR2 interactions, and are also shown to be potent inhibitors of RSV infection.
Definitions:
Peptides are defined herein as organic compounds comprising two or more amino acids covalently joined by peptide bonds. Peptides may be referred to with respect to the number of constituent amino acids, i.e., a dipeptide contains two amino acid residues, a tripeptide contains three, etc. Peptides containing ten or fewer amino acids may be referred to as oligopeptides, while those with more than ten amino acid residues are polypeptides. Such peptides may also include any of the modifications and additional amino and carboxy groups as are described herein.
Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
A (alanine)
R (arginine)
N (asparagine)
D (aspartic acid)
C (cysteine)
Q (glutamine)
E (glutamic acid)
G (glycine)
H (histidine)
I (isoleucine)
L (leucine)
K (lysine)
M (methionine)
F (phenylalanine)
P (proline)
S (serine)
T (threonine)
W (tryptophan)
Y (tyrosine)
V (valine)